Background: Pluripotency is a fundamental property of early mammalian development but it is currently unclear to what extent its cellular mechanisms are conserved in vertebrates or metazoans. POU5F1 and POU2 are the two principle members constituting the class V POU domain family of transcription factors, thought to have a conserved role in the regulation of pluripotency in vertebrates as well as germ cell maintenance and neural patterning. They have undergone a complex pattern of evolution which is poorly understood and controversial. Results: By analyzing the sequences of POU5F1, POU2 and their flanking genes, we provide strong indirect evidence that POU5F1 originated at least as early as a common ancestor of gnathostomes but became extinct in a common ancestor of teleost fishes, while both POU5F1 and POU2 survived in the sarcopterygian lineage leading to tetrapods. Less divergent forms of POU5F1 and POU2 appear to have persisted among cartilaginous fishes. Conclusions: Our study resolves the controversial evolutionary relationship between teleost pou2 and tetrapod POU2 and POU5F1, and shows that class V POU transcription factors have existed at least since the common ancestor of gnathostome vertebrates. It provides a framework for elucidating the basis for the lineage-specific extinctions of POU2 and POU5F1.